This invention relates, inter alia, to an assay method and to an assay device.
This invention relates in general to electrochemical assays. In such assays, the presence of an analyte of interest in a sample causes a measurable change in an electrochemical property of a sensor device. Typically, electrochemical assays are classified as xe2x80x9cpotentiometricxe2x80x9d or xe2x80x9camperometricxe2x80x9d, which measure changes in either potential or current respectively.
A number of electrochemical assays have been described. For example, enzyme electrodes have been used for the direct measurement of biomolecules such as glucose, urea, amino acids, and others in physiological samples. These enzyme electrodes include a selective enzyme layer immobilized at the surface of a potentiometric or amperometric device that senses the steady state concentration of a product formed in the immobilized layer as the substrate for the enzyme diffuses into this reactive film.
Other assays involve the use of Nafion(trademark) (E.I. Du Pont de Nemours and Co. Inc.) film. Nafion(trademark) is a polyanionic perfluorosulphonate ionomer with permselective properties large hydrophobic cations rather than small, hydrophilic cations tend to accumulate in the polymer via cationic exchange.
Limoges and Degrand (1993 Analytical Chemistry 65, 1054-1060) described a model system is which a Nafion(trademark)-coated electrode was used to detect the presence of amphetamine. The assay took the form of a competitive immunoassay, in which amphetamine in a sample competed with known amounts of labelled amphetamine for binding to amphetaminexe2x80x94specific antibodies. The competitor amphetamine was labelled with cobalticenium, which is a redox label.
The basis for the assay is that once bound by antibody, the labelled amphetamine is excluded from the Nafion(trademark) film because of the large size of the resulting amphetamine/antibody complex. Thus, in the presence of high concentrations of amphetamine in the sample, there is more free labelled amphetamine available for ion exchange and incorporation into the Nafion(trademark) film. Accordingly, the current corresponding to the oxidation or reduction of the cobalticenium-label in the film is proportional to the concentration of amphetamine in the introduced sample.
This sort of assay has several disadvantages and accordingly has not been widely adopted. In particular it requires the performance of a number of reaction steps before detection can effected by the Nafion(trademark)-coated electrode. The assay apparatus disclosed by Limoges and Degrand comprised a sensor which was not disposable or re-usable.
A different sort of electrochemical assay involves the use of an xe2x80x9cAntibody responsive membrane electrodexe2x80x9d, as described by Solsky and Rechnitz (1979 Science 204, 1308; 1981 Anal. Chim. Acta 123, 135). The antibody responsive membrane comprised ionophores (crown ethers) within a polyvinyl chloride matrix, the ionophores being conjugated to a hapten in such a way that the haptens projected from the surface of the membrane. The membrane was mounted in the tip of a conventional potentiometric membrane electrode. The addition of a sample containing antibodies specific for the hapten would allow antibodies to bind to the hapten which, in some unknown way, altered the electrochemical properties of the ionophores, changing the potential across the membrane.
The manner in which the device works is not understood, making it impossible rationally to design improvements thereon. Also, the associated detection system is large and cumbersome and not readily re-usable.
WO 89/11649 discloses a device for use in an electrochemical assay, the device comprising an electroactive polymer layer, within which layer are entrapped antibody molecules having binding specificity for an analyte of interest. Binding of the analyte of interest to the antibody inhibits the flow of counter ions from the environment surrounding the device into the space around the electroactive polymer, hence inhibiting electron flow to or from the polymer during a redox reaction. There is no disclosure of the electrochemical properties of the polymer being affected by the binding of a binding partner directly to the polymer.
WO 95/29199 discloses an electrode having a similar arrangement, wherein binding of a binding partner to a chemical moiety attached to an electroactive polymer can indirectly affect the electrochemical properties of the polymer. There is no disclosure of a binding partner having binding specificity for the electroactive polymer itself. A similar arrangement is disclosed in EP 0239969.
WO 93/25907 discloses a competition assay system involving an antigen of interest, and a derivatised antigen carrying a redox label, competing for binding to limiting amounts of antibody. Excess redox-labelled antigen is bound electrostatically to a polymer-coated film, so as to alter the redox potential across the film, which is measured in a conventional manner. The polymeric layer is not electroactive and essentially non-conducting.
EP 0402917 discloses a biosensor operating on a very similar principle to that disclosed in WO 89/11649: a conducting surface with an electroactive surfactant coating is modified by inclusion of one member of a specific binding pair. The analyte of interest is the other member of the specific binding pair. Binding of the reciprocal members of the specific binding pair blocks the movement of counter ions. There is no binding event involving binding directly to the electroactive surfactant.
WO 97/27474 discloses a method of determining the presence of an analyte of interest, whereby an electrode is coated with a member of a specific binding pair. In the absence of analyte of interest (which is the reciprocal member of the specific binding pair), an electroactive redox molecule can come into proximity with the electrode and donate electrons to, or accept electrons from, the electrode. This process is inhibited by the analyte of interest, which blocks the redox molecule from coming into proximity with the electrode. Thus there is no disclosure of the direct binding of a binding partner to an electroactive molecule so as to modify the electrochemical properties thereof. (All documents cited in the present specification are incorporated herein by reference).
The present invention aims to provide an improved type of electrochemical assay, particularly one which will be suitable for forming the basis of disposable, easy-to-use assay devices.
In a first aspect the invention provides a method of detecting the presence of an analyte of interest in a sample, the method comprising the steps of: providing an electrically conducting solid support having immobilised thereon a chemical moiety having an electroactive portion with an electrochemical property capable of being modulated in a detectable manner directly by the binding thereto of a binding partner having specific binding activity for the electroactive portion; and causing the binding partner to contact the electroactive portion of the chemical moiety as a result of the presence in the sample of the analyte of interest.
In a second aspect, the invention provides a component for a device for detecting the presence of an analyte of interest in a sample, the component comprising an electrically conducting solid support having immobilised thereon a chemical moiety, said chemical moiety comprising an electroactive portion with an electrochemical property capable of being directly modulated in a detectable manner by the binding thereto of a binding partner having specific binding activity for the electroactive portion.
The method of the invention may be used qualitatively, so as to indicate the presence or absence of the analyte of interest. Alternatively, and preferably, the method may be used quantitatively so as to indicate the amount (in relative or absolute units) of the analyte present. It will be apparent that all sorts of substances may be analytes of interest, although biological molecules (i.e. molecules present in or produced by living organisms) will typically be of most interest. These include nucleic acids (DNA and RNA or chimeras thereof, carbohydrates, enzymes, antigens, allergens), hormones (both protein and steroid varieties), especially sex and fertility hormones such as human chorionic gonadotrophin (hCG), estrone-3-glucuronide (E3G), progesterone-3-glucuronide (P3G), luteinising hormone (LH) and follicle stimulating hormone (FSH); and disease markers and diagnostic indicators (e.g. antibodies). Alternatively, the analyte of interest may be particulate e.g. bacterium, virus, yeast, fungus.
A particular advantage of the present invention is that it allows for assays to be conducted using samples which may be turbid, which samples can not be assayed using conventional colourimetric techniques. Examples of samples which are, or may be, turbid include: serum; whole blood; food and drink samples (e.g. milk); samples containing turbid growths of micro-organisms, and the like.
For the purposes of the present specification, the xe2x80x9celectroactive portionxe2x80x9d of the chemical moiety is defined as that portion which can donate and/or accept an electrical charge when the chemical moiety undergoes a redox reaction. Typically, the electrochemical property which is modulated in the assay will typically be one or more redox potentials. The electroactive portion may comprise one or more groups capable of undergoing oxidation or reduction under the assay conditions, the redox potential of any one or more of such groups being modulated by binding of the binding partner to the electroactive portion. Conveniently the one or more altered redox potentials may be detected by potentiometric or amperometric methods, known to those skilled in the art. Preferably amperometric detection methods (such as chronoamperometry) are employed, as these generate results which are generally easier to measure than potentiometric methods.
Preferably, the modulation of the electrochemical property of the electroactive portion is detected and/or measured by determining the amount of charge transferred to or from the electrically conducting solid support at a particular potential difference. The charge may be transferred between the electroactive portion and the electrically conducting solid support by movement of electrons, and/or ions, and/or other charged particles.
Conveniently, the electroactive portion of the chemical moiety, and the binding partner, are members of a specific binding pair. Advantageously the binding partner comprises an immunoglobulin or an effective portion thereof retaining specific binding activity for the chemical moiety. Effective portions of immunoglobulins therefore include, for example Fv, scFv, Fab, Fab2, and heavy chain variable regions (Hcv, such as those available from llamas and camels) or a chimeric molecule comprising any one or more of the aforementioned portions. Conveniently, the binding partner may be prepared by means of monoclonal antibody techniques or by selection and isolation from an appropriate library of nucleic acid sequences encoding binding partners (e.g. phage display libraries), which are both well known to those skilled in the art.
The electroactive portion of the chemical moiety is conveniently an intrinsic immunogen, or is at least capable of acting as an immunogen (i.e. is a hapten) when conjugated to appropriate carrier molecules (such as bovine serum albumin, or plant peptide derivative etc.). This facilitates the production of immunoglobulins (or effective portions thereof) having specific binding activity for the chemical moiety, which immunoglobulins may act as binding partners in the method of the invention.
In general, the chemical moiety will typically comprise as an electroactive portion an organic, moderately hydrophobic molecule. Examples include organometallic compounds (such as cobalticenium, and ferrocene), and heteroaromatic compounds (such as carbazoles, pyrroles, furans and thiophenes). Desirably the electroactive portion will typically comprise one or more groups readily capable of undergoing oxidation and/or reduction (oxidation being thought of as the removal of an electron, and reduction as the addition of an electron).
Oxidation or reduction will normally result in the creation of electrically charged entities, the effects of which may be stabilised by a conjugated system of electron orbitals in the moiety (xe2x80x9cdelocalised electronsxe2x80x9d). Accordingly, it is preferred that, in the conditions of the assay, the chemical moiety comprises a conjugated system of delocalised electrons. Particular examples of such chemical moieties include pyrroles, furans, thiophenes, and analogues and/or multimers thereof (as described, for example, by Diaz et al., 1979 J. Chem. Soc. Chem. Commun. p635; Niziurski-Mann et al., 1993 J. Am. Chem. Soc. 115, 887; and Waltman et al., 1984 J. Phys. Chem. 88, 4343). The non-bonding electrons of the respective Nitrogen, Oxygen and Sulphur atoms contribute to the conjugated system of electron orbitals. A preferred moiety is N-alkyl carbazole or analogues thereof (in monomeric, dimeric or polymeric forms). FIGS. 1A-D show the general structure of pyrrole, furan, thiophene and carbazole monomers, respectively.
In general it will be desired for the chemical moiety to be immobilised upon a solid support (e.g. an assay dipstick, or a capillary fill chamber). Preferably the chemical moiety will be immobilised upon an electrically conducting portion of the solid support, which electrically conducting portion may comprise, for example, a thin strip of gold, platinum or other conducting metal, a metal oxide, carbon/graphite, silicon, or silicate.
The immobilisation of the chemical moiety upon the solid support may be accomplished in several ways. The present inventors have found that one method is to place a solid support in a solution of a suitable chemical moiety precursor, and then to cause in situ formation of the chemical moiety upon the solid support. Typically, such a process may take the form of causing polymerisation of precursor monomers upon the solid support, resulting in the formation of a mesh-like coating of chemical moiety upon the solid support.
Alternatively, and more preferably the chemical moiety may be immobilised upon the solid support by means of an intervening xe2x80x9cpendantxe2x80x9d chain portion, preferably one which allows for self-assembly as a monolayer on a supporting surface (as described, for example, by Sabatini and Rubinstein, 1987 J. Phys. Chem. 91, 6663-6669; von Velzen et al., 1994 J. Am. Chem. Soc. 116, 3597-3598; Chidsey et al., 1990 J. Am. en. Soc. 112, 4301-4306; and Rubinstein et al., 1988 Nature 332, 426-429). The chain portion may be thought of as forming an integral part of the chemical moiety.
The use of a pendant portion or molecule is preferred as it separates the electroactive portion from the electrically conducting surface of the electrode (whilst preferably, but not necessarily, retaining a degree of electrical conductivity through, for example a system of delocalized electrons). The pendant portion is typically substantially linear. A preferred pendant portion is an alkyl or alkenyl group (typically comprising 3 to 14, preferably 5 to 12, carbon atoms), which may be substituted or unsubstituted.
In a particular embodiment, an alkyl or alkenyl pendant group is attached to the electrode via a sulphydryl or thiol groupxe2x80x94other chemical attachments may be equally suitable.
Another advantage of the use of pendant groups or portions is that they tend to fill in xe2x80x9cpin holesxe2x80x9d (minor irregularities in the surface of the conducting layer of the electrode), which can have a detrimental effect on the reproducibility of results obtained. It may also be preferred to include excess pendant molecules attached to the electrode (i.e. not every pendant group will necessarily be joined to an electroactive portion). The presence of excess pendant groups is throught to improve the stability and rigidity of the monolayer, which optimises the method/device of the invention. In particular embodiments, the inventors have found that a ratio of four pendant portions to three electroactive portions, may provide optimum results. The excess pendant molecule need not be identical to the pendant portion attached to the electroactive portion: thus, excess pendant xe2x80x9cspacerxe2x80x9d molecules may be deliberately introduced. Such spacer molecules will conveniently be of a similar nature to the pendant portion of the chemical moiety (e.g. alkyl or alkenyl), but will be no longer in chain length, and possibly shorter, than the pendant portion of the chemical moiety, so as to avoid the possibility that the spacer molecules cause steric hindrance when the binding partner attempts to bind to the electroactive portion.
It is highly preferred that the assay method is such that the analyte of interest need not be identical to the electroactive portion of the chemical moiety to which the binding partner binds. In this way, the assay method may be adapted to detect the presence of any analyte of interest, with the binding partner (typically immunoglobulin) specific for the electroactive portion being required simply as the last step of the assay to generate a detectable signal by binding to the electroactive portion. This preferred feature may be obtained, for example, by utilising competition or displacement type methodologies, as will be explained below.
Accordingly, in preferred embodiments, the binding partner is present as a binding entity, which binding entity comprises first and second specific binding activities. The first specific binding activity is for the electroactive portion of the chemical moiety as aforesaid. The second specific binding activity is typically for the analyte of interest, or for a molecule (such as an immunoglobulin) which itself has specific binding activity for the analyte of interest, such that the presence of the analyte of interest tends to displace the binding entity from a solid support. The binding entity may also possess further specific binding activities, but these are not essential to performance of the invention.
Conveniently the binding entity will be a bispecific antibody or xe2x80x9cDiabodyxe2x80x9d or other bispecific immunoglobulin fragment (e.g. double headed scFv, double headed HCV or a chimeric molecule comprising an scFv and/or an HCV fragment). Alternatively, the binding entity may comprise a non-binding component, to which are attached first and second binding partners having respective first and second specific binding activities. These binding partners may comprise conventional immunoglobulin molecules, such as monospecific antibodies, or effective binding portions thereof (e.g. scFv etc.). The non-binding component may be any substance large enough, and with appropriate chemical properties, for the first and second binding partners to be attached thereto. The non-binding component may be, for example, a peptide, a polypeptide, a liposome or, more conveniently, a particle such as a latex bead. Methods of attachment of immunoglobulins to latex beads are well-known to those skilled in the art.
Conveniently, the method step of causing the binding partner to contact the electroactive portion as a result of the presence of the analyte of interest in the sample is effected by the analyte causing displacement or release of the binding partner from a solid support to which the binding partner is releasably immobilised prior to introduction of the sample. The analyte will typically displace the binding partner from, or compete with the binding partner for binding to, binding sites on the solid support by means of which the binding partner is releasably immobilised.
Thus, in particular embodiments, the assay method involves use of a first solid support upon which is immobilised the chemical moiety, and a second solid support (which may be a separate portion of the first solid support, or a discrete component) upon which is immobilised an analogue of the analyte of interest. The analogue is such that the second specific binding activity of the binding entity will bind to the analogue, albeit with lower affinity than for the analyte of interest. Accordingly, prior to performance of the assay, the binding entity is reversibly immobilised upon the second solid support via its second specific binding activity.
In the presence of free analyte of interest, the analyte will compete with the immobilised analogue for binding to the binding entity via the second specific binding activity. Typically, the affinity of the binding entity for the analyte is greater than that for the analogue, such that the binding entity will be displaced from the second solid support if the analyte of interest is present in the sample. The displaced binding entity is then free to react, via its first specific binding activity, with the electroactive portion of the chemical moiety immobilised on the first solid support, thereby modulating an electrochemical property of the electroactive portion in a detectable manner as aforesaid.
Accordingly, in preferred embodiments, there is an affinity difference between the binding affinity of the analogue and the analyte of interest respectively, such that the presence of the analyte, even at low concentration, will tend to displace the binding entity from the analogue. Alternatively there may be no difference in binding affinity, and the binding entity is displaced from the analogue by competition, e.g. because the analyte of interest is present in the sample at a greater effective concentration than the analogue.
The displaced binding entity may be allowed to diffuse from the second solid support to the first solid support, especially where the first and second supports are in close proximity (e.g. 50 xcexcm-5 mm, preferably 50 xcexcm-1 mm). Alternatively, the binding entity may be transported by capillary action along or through a porous medium, or may be transported by a flow of a fluid sample (e.g. a body fluid such as blood or urine and the like), as disclosed in, for example, WO 91/05262. A pump means (e.g. syringe pump or peristaltic pump) may be provided, if appropriate, to pump fluid comprising released binding entity from the second solid support to the first solid support.
Use of analogues of analytes of interest, in a slightly similar manner, is disclosed and taught, for example, in EP 0 324 540, and in PCT/EP95/04518. Those skilled in the art will appreciate that the analyte of interest will typically be a biological molecule, such as a peptide or polypeptide, or a steroid hormone or the like. Conveniently, the analogue of the analyte of interest will be an epitope mimic, i.e. a molecule (typically smaller than the analyte of interest) generally of synthetic origin, such as a short peptide, which behaves in a manner comparable to the binding site of the analyte to which the binding partner binds. Examples of analytes and suitable analogues are disclosed in EP 0 324 540 and PCT/EP95/04518. The embodiment described above is a variant of known displacement/competition type assays, disclosed inter alia in WO 91/05262 and EP 0 383 313.
In a further embodiment the binding entity is such that the second specific binding activity is for an antibody directed against the analyte of interest. The binding entity may comprise, for example, an analogue of the analyte of interest, which analogue is relatively loosely bound by antibody specific for the analyte of interest, which antibody is immobilised on the second solid support. Alternatively the binding entity may comprise an anti-idiotypic antibody specific for the binding site of the immobilised antibody. In any event, the immobilised antibody conveniently (but not essentially) has a greater affinity for the analyte than for the binding entity, such that the presence of the analyte of interest in the test sample will tend to cause the displacement of the binding entity from the second solid support. The displaced binding entity is then free to bind to the first solid support, via its first specific binding activity, as outlined above.
Methods of immobilising antibodies on solid supports are known to those skilled in the art. Useful discussion is provided by G Hermanson, in xe2x80x9cBioconjugate Techniquesxe2x80x9d (Academic Press, 1996). Typically the immobilised antibody is covalently coupled by an added functional group. Conveniently the antibody for the analyte may be immobilised by attachment to a further antibody-specific antibody immobilised on the solid support. The first solid support will typically comprise an electrode. The second solid support may be any of those routinely used in assays and include, for example, synthetic plastics materials, microtitre assay plates, latex beads, filters, glass or plastics slides, dipsticks etc. Advantageously, the second solid support comprises a wettable surface.
Conveniently the component of the second aspect of the invention is used to perform an assay of the type disclosed in our co-pending European patent application No. 97309409.7, filed on Nov. 21, 1997. The component might take the form of a relatively cheap, disposable or replacable part for use and interaction with other components (e.g. a separate signal detection means, acting as a reader for the assay result). Alternatively, the component may be provided as an integral part of a larger device.
In a third aspect, the invention provides an assay device comprising the component of the second aspect. Conveniently the device further comprises one or more of the following: sample receiving means for accepting a sample under test; a binding partner having specific binding activity for the electroactive portion of the chemical moiety; detection means for detecting a modulation in an electrochemical property of the electroactive portion of the chemical moiety; data processing means for processing data output from the detection means; and data display means for displaying the assay result, preferably in a numerical form.
The assay device will conveniently comprise a capillary-fill reaction chamber as part of the sample receiving means. In a preferred embodiment the chamber is at least partly defined by the first and second solid supports of the preferred method aspect. Capillary-fill devices which may be adapted for use in the present invention are taught, for example, in U.S. Pat. No. 5,141,868.
In a fourth aspect the invention provides a component for use in the device defined above, said component comprising a solid support having releasably immobilised thereon a binding partner having specific binding activity for an electroactive portion of a chemical moiety, the binding partner being displaced from the solid support in the presence of the analyte of interest, and wherein binding of the binding partner to the electroactive portion directly modulates an electrochemical property of the electroactive portion in a detectable manner. As with the component of the second aspect of the invention, the component may be provided as a relatively cheap, disposable or replacable part for use and interaction with other components (e.g. a separate signal detection means, acting as a reader for the assay result), or may be provided as an integral part of a larger device.
In a fifth aspect, the invention provides a chemical moiety, comprising an electroactive portion, having the structure shown in FIG. 13 wherein: R1 and R2 are independently H; OH; C1-C14 alkyl, aryl, alkenyl or alkoxy (all optionally substituted); halide; amide; or amine; and further wherein the heteroaromatic ring structure may be optionally substituted at one or more positions with alkyl, aryl, alkenyl, or alkoxy groups (all themselves optionally substituted), acid groups (organic or inorganic), halide, amide or amine.
Advantageously R1 is alkyl, preferably ethyl, propyl or butyl and preferably R2 is C1-C12 alkyl, more preferably C4-C8 alkyl, and most preferably C6 alkyl. It is generally preferred that R1 and R2 are not identical. In a preferred embodiment R2 comprises a (preferably terminally positioned) reactive substituent such as a thiol, carboxyl, amide, amine, halide, aldehyde, ketone, epoxide, or succinimide group, or other protein coupling agent (e.g. as mentioned in Hermanson, xe2x80x9cBioconjugate Techniquesxe2x80x9d, Academic Press, 1996) which facilitates coupling of the moiety to other entities (e.g. solid surfaces). In a preferred embodiment, the chemical moiety is 3,3(N-[6-thiol hexyl]carbazole)N-ethyl carbazole.
The chemical moiety of the fifth aspect of the invention may conveniently be immobilised on an electrically conducting solid support, so as to form a component in accordance with the second aspect of the invention.
In a sixth aspect, the invention provides a molecule having binding specificity for the chemical moiety of the fifth aspect defined above. More particularly the molecule preferably has binding specificity for the electroactive heteroaromatic ring portion of the chemical moiety. The molecule will conveniently comprise an immunoglobulin molecule or an effective portion thereof which retains binding specificity for the chemical moietyxe2x80x94such portions include, for example, Fv scFv, Fab, Fab2, HCV, or a chimeric molecule comprising any one or more of the aforementioned portions. In a preferred embodiment the molecule will comprise at least two binding specificities: a first binding specificity for the chemical moiety, as aforementioned; and a second binding specificity for an analyte of interest or for an antibody (or effective antigen-binding portion thereof) directed against an analyte of interest. It will be apparent to the reader that molecules in accordance with the sixth aspect of the invention will conveniently be suitable for use as a binding partner in performing the method of the first aspect and/or may conveniently be releasably immobilised on a solid support so as to provide a component in accordance with the fourth aspect of the invention.